The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose-grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose- and lactate-grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections.

Uridine auxotrophy, based on disruption of both URA3 alleles in diploid Candida albicans strain SC5314, has been widely used to select gene deletion mutants created in this fungus by "Ura-blasting" and PCR-mediated disruption. We compared wild-type URA3 expression with levels in mutant strains where URA3 was positioned either within deleted genes or at the highly expressed RPS10 locus. URA3 expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5-monophosphate decarboxylase. Reduced URA3 expression following integration at the GCN4 locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (URA3) and CAI-4 (ura3) proteomes revealed that inactivation of URA3 caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of URA3 at the RPS10 locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in C. albicans can be associated with the selectable marker rather than the target gene. Reintegration of URA3 at an appropriate expression locus such as RPS10 can offset most problems related to the phenotypic changes associated with gene knockout methodologies.Genes encoding enzymes in the uridine biosynthetic pathway are used as positive selectable markers in gene disruption strategies for a wide range of eukaryotic organisms. For the major human pathogenic fungus Candida albicans, this approach, based on the URA3 gene, was the only means available for gene disruption for more than 10 years. C. albicans is diploid and has no known natural sexual cycle that leads to a haploid form. Therefore, molecular genetic evidence associating phenotypic changes with individual gene functions requires the successive disruption of two copies of the gene of interest. "Ura-blasting," used for gene disruption in C. albicans (28), is based on a similar approach in Saccharomyces cerevisiae (1) and makes use of a C. albicans ura3 auxotroph. Transformants are selected on the basis of uridine prototrophy. The marker is then recycled by use of 5-fluoroorotic acid to select for Ura Ϫ segregants that have lost URA3 through a reciprocal crossover between identical flanking DNA repeats. The "Ura-blaster" can then be used to disrupt the second allele in these ura3 segregants. PCR-based gene disruption protocols also exploit the URA3 marker but often do not recycle it (90).One area of investigation that has made extensive use of specific gene disruption based on the URA3 selection strategy is the study of virulence in C. albicans. At least 50 genes have been implicated as putative virulence factors in this fungus based on results of mouse intravenous challenge experiments in which the homozygous C. albicans null mutants of interest were shown to be less lethal than wild-type controls (61). In most o...

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Candida albicans is the major fungal pathogen of humans. This opportunistic pathogen can cause irritating superficial infections of the mucosa and serious life threatening systemic infections in the immunocompromised patient (1, 2). Invasive candidosis in hospitals now represents the third or fourth most common form of septicaemia (3, 4). The cell surface of C. albicans is the immediate point of contact between the fungus and host and plays vital roles in adhesion and immunomodulation of host responses, and it is a source of antigens (5-8). The outer cell wall layer is enriched in mannoproteins, which are embedded in a matrix of structural polysaccharides consisting of ␤-1,3-and ␤-1,6-linked glucan and chitin (9). This layer is important in adhesion to host surfaces and their subsequent colonization (10 -12). Both the protein and carbohydrate components of mannoproteins have been implicated in adhesion to the host (10, 13-15), although details of the nature of the ligands and receptors are still lacking. Hence, glycosylation of cell wall proteins is critical for host-fungal interactions and pathogenicity. Mnt2p also functions inKnowledge of glycosylation in Saccharomyces cerevisiae (16 -28) and information from the C. albicans genome data base has provided significant resources for the identification and analysis of glycosylation genes in C. albicans. Mannoproteins of S. cerevisiae and C. albicans contain both N-and O-linked oligosaccharides. The N-linked glycans, attached to asparagine residues of proteins, contain a conserved core structure and an elaborate, highly branched outer mannose chain that is specific to fungi and contains both acid-stable and acid-labile components (17,29,30). Glycosylation in C. albicans has its own relevance in investigations of the role of specific oligosaccharide moieties in host-fungal interactions. The acid-labile mannosylphosphate component, containing ␤-1,2-linked mannose, has been implicated in adhesion and recognition of phagocytic leukocytes, although mutants lacking this component have been shown to have normal interactions with macrophages (31). Both ␤-1,2-and ␣-1,2-linked mannan oligosaccharides have been implicated directly in adhesion functions (12,32).In C. albicans, O-glycans are linear oligosaccharides of one to five ␣-1,2-linked mannose residues (32-34). In S. cerevisiae an ␣-1,2-linked O-linked glycan is capped with one or two ␣-1,3-linked mannose residues (27). O-Glycosylation in S. cerevisiae is initiated in the endoplasmic reticulum where at least four of the seven-membered PMT gene family act to transfer mannose from dolichyl phosphate-activated mannose to serine or threonine (18,35,36). Evidently this step is essential, as certain combinations ...

SummaryEukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca2+ cytoplasmic gradient, which is correlated with their ability to exhibit polarized tip growth and to respond to growth-directing extracellular signals [1–5]. In yeast and other fungi, the polarisome, exocyst, Arp2/3, and Spitzenkörper protein complexes collectively orchestrate tip growth and cell polarity, but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic Ca2+ gradients. Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) [6] and imposed electric fields (galvanotropism) [7]. The establishment and maintenance of directional growth in relation to these environmental cues was Ca2+ dependent. Tropisms were attenuated in media containing low Ca2+, or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca2+ influx at sites of polarized growth via Ca2+ channels that are activated by appropriate environmental signals.

ThecellwallproteinsoffungiaremodifiedbyN-andO-linkedmannosylation and phosphomannosylation, resulting in changes to the physicalandimmunologicalpropertiesofthecell.Glycosylationofcell wall proteins involves the activities of families of endoplasmic reticulum and Golgi-located glycosyl transferases whose activities are difficult to infer through bioinformatics. The Candida albicans MNT1/ KRE2 mannosyl transferase family is represented by five members. We showed previously that Mnt1 and Mnt2 are involved in O-linked mannosylation and are required for virulence. Here, the role of C. albicans MNT3, MNT4, and MNT5 was determined by generating single and multiple MnT⌬null mutants and by functional complementation experiments in Saccharomyces cerevisiae. CaMnt3, CaMnt4, and CaMnt5 did not participate in O-linked mannosylation, but CaMnt3 and CaMnt5 had redundant activities in phosphomannosylation and were responsible for attachment of approximately half of the phosphomannan attached to N-linked mannans. CaMnt4 and CaMnt5 participated in N-mannan branching. Deletion of CaMNT3, CaMNT4, and CaMNT5 affected the growth rate and virulence of C. albicans, affected the recognition of the yeast by human monocytes and cytokine stimulation, and led to increased cell wall chitin content and exposure of ␤-glucan at the cell wall surface. Therefore, the MNT1/KRE2 gene family participates in three types of protein mannosylation in C. albicans, and these modifications play vital roles in fungal cell wall structure and cell surface recognition by the innate immune system.The human pathogen Candida albicans is the most frequent cause of systemic candidosis, which is a common, life-threatening infection in immunocompromised patients (1). The C. albicans cell wall is a robust yet dynamic structure that protects the cell from changes in the extracellular environment. It is the immediate contact point with host cells and contains antigenic determinants, glycoproteins involved in the adhesion to host tissues, and most of the pathogen-associated molecular patterns that are recognized by host immune system (2). The wall is organized in an inner skeletal layer comprising chitin, ␤1,3-and ␤1,6-glucans, and an outer layer that is dominated by highly glycosylated proteins (3). These proteins are post-translationally modified with N-and/or O-linked mannans, both of which can be further elaborated with oligomannosides that are attached via phosphodiester linkages (phosphomannans). Mannans have important roles in cell wall integrity, adhesion to host cells and tissues, virulence, and the establishment of a response by immune cells (2, 4 -10). The O-and N-linked mannans, along with ␤-glucans, represent the main C. albicans pathogen-associated molecular patterns recognized by the innate immune system (2, 11-13).Mannan biosynthesis has been carefully characterized in Saccharomyces cerevisiae, and the main features of the pathways involved in the construction of these oligosaccharides are conserved in C. albicans. However, N-and O-linked mannans of C. al...

Most of the fungal species that infect humans can grow in more than one morphological form but only a subset of pathogens produce filamentous hyphae during the infection process. This subset is phylogenetically unrelated and includes the commonly carried yeasts, Candida albicans, C. dubliniensis, and Malassezia spp., and the acquired pathogens, Aspergillus fumigatus and dermatophytes such as Trichophyton rubrum and T. mentagrophytes. The primary function of hypha formation in these opportunistic pathogens is to invade the substrate they are adhered to, whether biotic or abiotic, but other functions include the directional translocation between host environments, consolidation of the colony, nutrient acquisition and the formation of 3-dimensional matrices. To support these functions, polarised hyphal growth is co-regulated with other factors that are essential for normal hypha function in vivo.

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.

Hypha orientation is an essential aspect of polarised growth and the morphogenesis, spatial ecology and pathogenesis of fungi. The ability to re-orient tip growth in response to environmental cues is critical for colony ramification, the penetration of diverse host tissues and the formation of mating structures. Recent studies have begun to describe the molecular machinery regulating hypha orientation. Calcium signalling, the polarisome Bud1-GTPase module and the Tea cell-end marker proteins of the microtubule cytoskeleton, along with specific kinesins and sterol-rich apical microdomains, are involved in hypha orientation. Mutations that affect these processes generate normal-shaped, growing hyphae that have either abnormal meandering trajectories or attenuated tropic responses. Hyphal tip orientation and tip extension are, therefore, distinct regulatory mechanisms that operate in parallel during filamentous growth, thereby allowing fungi to orchestrate their reproduction in relation to gradients of effectors in their environments.